Pharmaceutical lysine-containing polypeptide compositions and methods of use thereof

ABSTRACT

Pharmaceutical compositions and methods are provided for the therapy of immunodeficient, immunodepressed or hyperactive immune states and for the prevention and treatment of opportunistic infections in such states comprising administering to a subject a pharmaceutically acceptable composition comprising as an active ingredient peptides having the formula R′-L-Glx-L-Glx-L-Lys-R″ and/or their pharmaceutically acceptable salts; wherein Glx is Gln or Glu.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

[0001] This is a continuation-in-part of (1) copending Ser. No.07/967,633, filed Oct. 28, 1992; (2) copending Ser. No. 07/783,517,filed Oct. 28, 1991; and (3) copending Ser. No. 07/816,205, filed Jan.2, 1992; each of which are incorporated herein by reference.

[0002] The present invention is directed to peptide pharmaceuticalcompositions and uses thereof, in particular, small peptides includingthe amino acid sequence, Glx-Lys. These peptide compositions are usefulfor modulation of the immune system; the treatment of immunodepressedstates and of opportunistic infections in immunodepressed statesassociated with acquired immune deficiency syndrome; treatment ofinfections caused by bacterial, viral, fungal, and parasitic organisms;augmentation of vaccination response; treatment of atopic states,treatment of anemias; treatment of leukocytic disorders; and the like.

BACKGROUND OF THE INVENTION

[0003] A wide variety of diseases are caused by abnormalities of theimmune system and hematological systems of animals including man.Further, the immune system is required to treat many other diseases. Forexample, immunodeficiencies such as the acquired immunodeficiencysyndrome (AIDS) most commonly cause premature death and disability bymaking the person susceptible to infections and malignancies. Thissusceptibility is caused by a weakening of the immune system.Specifically, the HIV virus that causes AIDS attacks and kills T helpercells. Loss of T helper cells compromises the host's ability to fightinfections by normal immunological mechanisms. Many types of malignancyare also associated with immunodeficient states suggesting that they arecaused by a failure of normal host immune surveillance systems. Manydiseases cause secondary immunodeficiency allowing for more rapidprogression of the primary disease or the development of secondarydiseases. Methods to enhance suppressed immune systems have not achievedhigh levels of efficacy.

[0004] Diseases may also be caused by hyperactivity of the immunesystem. For example, collagen vascular diseases are associated withimmunologically-mediated damage to the host tissue. Such diseasesinclude multiple sclerosis, rheumatoid arthritis and the like. Thesediseases afflict many individuals and cause significant morbidity andmortality. Treatments generally include immune suppression.Unfortunately, generalized immune suppression often results in increasedincidence of infections and malignancies. Therefore, to treat onedisease patients are placed at risk for developing other, possibly lifethreatening, diseases.

[0005] Immunological stimulation, even in healthy individuals, may aidin the treatment of several diseases. Infectious diseases may be moreeffectively treated by stimulation of the immune system. The enhancedimmunological response may work with other treatments to eliminate theinfection more readily. Also, specific immune stimulation has been shownto reduce tumor size in some malignancies. Also, many drugs used forprimary treatment of infections and malignancies have significant sideeffects. Therefore, it is desirable to reduce the dose of the primarydrug whenever possible.

[0006] What is needed in the art are compositions and methods formodulating the immune system. Ideally, these compounds and methods wouldbe able to stimulate suppressed or deficient immune systems as well asreduce immune hyperactivity. Also these compounds and methods should actto restore a natural balance to the immune system. Quite surprisingly,the present invention fulfills these and other related needs.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention provides compositions comprising a peptidehaving the formula R′-Glx-Glx-Lys-R″ or a pharmaceutically acceptablesalt thereof; wherein Glx is Glu or Gln; R′ is H- or a first amino acidsequence having fewer than 7 amino acids; R″ is -H or a second aminoacid sequence having fewer than 7 amino acids; and the peptide has asequence of at least 5 and not more than 9 amino acids. Generally, R′ isH-, Thr-Ala-, Thr-Pro-, Ser-Ala-, Ser-Pro-, Ser-Ser-, Met-Leu-Thr-Ala-,or Leu-Thr-Ala-; and R″ is -H, -Ala, -Ala-Ala or -Ala-Val. In preferredembodiments, the peptide is L-Thr-L-Pro-L-Glu-L-Glu-L-Lys orL-Thr-L-Ala-L-Glu-L-Glu-L-Lys.

[0008] Also provided are pharmaceutical preparations comprising apeptide having the formula R′-Glx-Lys-R″ or a pharmaceuticallyacceptable salt thereof, wherein Glx is Glu or Gln; R′ is H- or a firstamino acid sequence having fewer than 7 amino acids; R″ is -H or asecond amino acid sequence having fewer than 7 amino acids; and thepeptide has a sequence of at least 2 and not more than 9 amino acids;and a physiologically acceptable carrier. In preferred embodiments, thepeptide is L-Glu-L-Lys, L-Thr-L-Ala-L-Glu-L-Glu-L-Lys² orL-Thr-L-Pro-L-Glu-L-Glu-L-Lys.

[0009] Methods for using the peptides of the present invention are alsoprovided. These methods include administration of the pharmaceuticalpreparations of the present invention for immunomodulation of a host'simmune system, treatment of infections, treatment of anemias, treatmentof atopic states, and treatment of leukocytic disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 illustrates an experimental method for assessing treatmentof infection with EK.

[0011]FIG. 2 illustrates survival of bacterially-infected mice treatedwith EK with and without an adjuvant antibiotic.

[0012]FIG. 3 illustrates survival of bacterially-infected mice treatedwith EK without adjuvant antibiotics.

[0013]FIG. 4 illustrates survival of bacterially-infected mice treatedwith EK with adjuvant antibiotics.

[0014]FIG. 5 illustrates an experimental protocol to assess the efficacyof HM897 in the treatment of bacterial infection in mice.

[0015]FIG. 6 illustrates survival of bacterially-infected mice treatedwith HM897 with and without adjuvant antibiotics.

[0016]FIG. 7 illustrates survival of bacterially-infected mice treatedwith HM897 with adjuvant antibiotics.

[0017]FIG. 8 illustrates survival of bacterially-infected mice treatedwith HM897 without adjuvant antibiotics.

[0018]FIG. 9 illustrates survival of vaccinated fish treated withdifferent amounts of HM897.

[0019]FIG. 10 illustrates survival of unvaccinated fish treated withdifferent amounts of HM897.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is based in part on the discovery thatcertain peptide compositions exhibit a broad range of efficacy formodulation of the immune system. This provides a means for theprevention and treatment of infections in immunocompetent as well asimmunodepressed states, and for therapeutically effective treatment ofimmunodeficient states, particularly AIDS. Other disorders associatedwith immune and hematologic systems may be similarly treated. This isbelieved to be highly unexpected for such relatively small compounds toexhibit such a broad range of activity. Furthermore, we have not foundany significant side effects from the use of the peptides according tothe present invention. Due to their simple nature, the peptides of thepresent invention are relatively inexpensive to manufacture.

[0021] The present invention provides peptide compositions,pharmaceutical preparations containing the peptides, and methods fortherapeutic use of the peptides. Generally, the compositions comprise apeptide having the formula R′-Glx-Glx-Lys-R″ or a pharmaceuticallyacceptable salt thereof; wherein Glx is Glu or Gln; R′ is H- or a firstamino acid sequence having fewer than 7 amino acids; R″ is -H or asecond amino acid sequence having fewer than 7 amino acids; and thepeptide has a sequence of at least 5 and not more than 9 amino acids.

[0022] The pharmaceutical preparations of the present inventiongenerally comprise a peptide having the formula R′-Glx-Lys-R″ or apharmaceutically acceptable salt thereof, wherein Glx is Glu or Gln; R′is H- or a first amino acid sequence having fewer than 7 amino acids; R″is -H or a second amino acid sequence having fewer than 7 amino acids;and the peptide has a sequence of at least 2 and not more than 9 aminoacids; and a physiologically acceptable carrier. The peptides andpharmaceutical preparations may be employed in a variety of therapeuticuses. These include modulating the activity of a host's immune system,treating infections in a host, treating atopic states, treatingleukocytic disorders, treating anemias in a host, and augmentingvaccination responses.

[0023] As used herein, the terms “immunomodulator” and“immunomodulating” encompass the activity of restoring the naturalbalance to a host's immune system. This includes enhancing or restoringthe subject's immune system, as evidenced by measurable blood parametersand/or the patient's improved ability to combat infection or disease,and the ability to heal tissue. Hence, immunomodulation encompassesimprovement of the immune system due to an immunodeficient state (forexample, caused by removal of the thymus), and/or an immunodepressedstate (for example, caused by exposure to radiation). Furthermore, thepresent invention provides for modulation of the immune by loweringblood parameters and other indicia of the immune state if these indiciaare abnormally elevated. The present invention encompasses thetherapeutic method of treating the immunodeficient, immunodepressed orelevated immune state per se, thus providing prophylaxis againstinfection and disease, as well as a treatment of infection, disease orwound by enhancing the immune system.

[0024] Generally, the peptide will have formula of Formula I, using thenormal convention wherein the first named amino acid is the aminoterminus and the last named amino acid is the carboxyl terminus.

R′-Glx-Lys-R″  (I)

[0025] wherein:

[0026] R′ is H-, Thr-Ala-Glx-, Thr-Pro-Glx-, Ser-Ala-Glx-, Ser-Pro-Glx-,Ser-Ser-Glx-, Met-Leu-Thr-Ala-Glx-, or Leu-Thr-Ala-Glx-;

[0027] R″ is -H, -Ala, -Ala-Ala or -Ala-Val; and

[0028] Glx is Glu or Gln.

[0029] In accordance with a preferred embodiment of the presentinvention are pharmaceutical preparations comprising of the Formula II(Formula I wherein R″=H);

R′-Glx-Glx-Lys  (II)

[0030] wherein:

[0031] R′ is Thr-Ala-Glx-, Thr-Pro-Glx-, Ser-Ala-Glx-; Ser-Pro-Glx-, orSer-Ser-Glx-.

[0032] Preferred species are Glx-Lys and Thr-Ala-Glx-Glx-Lys,particularly wherein Glx=Glu. The amino acids of the peptides of thepresent invention may be either D or L stereoisomers. The amino acids ina peptide may all be either L or D or a mixture of L and Dstereoisomers. It is generally preferred that all of the amino acids beof the L form. Specific amino acid stereoisomers will be denoted by aprefix of L- or D-. For example, the L stereoisomer of alanine isdenoted L-Ala.

[0033] Species in which R″=H indicates a free C-terminus carboxyl group.

[0034] Other particularly preferred species useful in accordance withthe invention are the peptides according to Formula I wherein:

R′=Glx-

R′=Thr-Pro-Glx-

R′=Met-Leu-Thr-Ala-Glx- and R″=-Ala;

R′=Leu-Thr-Ala-Glx- and R″=-Ala;

R′=Leu-Thr-Ala-Glx- and R″=-Ala-Ala;

R′=Leu-Thr-Ala-Glx- and R″=-Ala-Val.

[0035] The peptides of the present invention may be combined inpharmaceutical preparations for a variety of therapeutic uses. Thepreparations may be administered to a variety of hosts for therapeuticpurposes. Suitable hosts include human and non-human primates, domesticanimals including dogs, cats, rodents, birds, horses, cows, pigs, fish,and the like.

[0036] The compositions may also find use for pre- or post-exposureprophylaxis, e.g., HIV prophylaxis following “dirty needle” injuries tohealth care workers or routinely accompanying blood transfusions or topersons in danger of becoming exposed to infected body or culturefluids. The peptides of the present invention are particularly usefulfor augmentation of vaccinations. By “augmentation of vaccines”, it ismeant that the level and/or duration of complete or partial protectionfrom disease obtained from vaccination is enhanced.

[0037] Administration of the peptides of the present invention isconjunction with a vaccine may enhance the immune response to thevaccine providing both a higher level of immunity and a prolongedanamnestic response. The peptides may be administered prior to,simultaneously with, or following vaccination. Generally, the peptideswill be administered prior to or simultaneously with vaccination.

[0038] The pharmaceutical compositions are intended for parenteral,topical, oral, or local administration for prophylactic and/ortherapeutic treatment. Preferably, the peptides of the present inventionare administered intramuscularly or intranasally. As the peptides of thepresent invention may be administered parenterally, i.e., intravenously,subcutaneously, intramuscularly, or intrathecally, this inventionprovides pharmaceutical preparations for parenteral administration whichcomprise a solution of a peptide of the present invention dissolved in apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, e.g., water, buffered water,0.4% saline, 0.3% glycine, and the like, including glycoproteins forenhanced stability, such as albumin, lipoprotein, globulin, etc. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The resulting aqueous solutions may be packaged for use orfiltered under aseptic conditions and lyophilized, the lyophilizedpreparation being combined with a sterile aqueous solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0039] The active peptides of the pharmaceutical preparations accordingto the present invention may be used as free peptides or in the form ofa water soluble pharmaceutically acceptable salt, such as a sodium,potassium, ammonium or zinc salt. In addition to the peptides andphysiologically acceptable carriers, the pharmaceutical preparations mayinclude other active ingredients which independently impart an activityto the composition, such as antibiotics, interferon, anesthetics, andthe like.

[0040] The concentration of the peptides of the present invention inthese pharmaceutical preparations can vary widely, i.e., from about0.001% to as much as 15 or 20% by weight and will be selected primarilyby fluid volumes, viscosities, etc., in accordance with the particularmode of administration selected. When utilized intramuscularly as aninjection solution with the active ingredient in a therapeuticallyeffective immunopotentiating amount of about 0.001 to 0.01% by weight.If prepared in the form of a tablet, capsule or suppository, it ispreferred that the active ingredient be present in an amount of about0.1 mg per tablet, suppository or capsule. In such form, the capsule,suppository or tablet may also contain other conventional excipients andvehicles such as fillers, starch, glucose, etc. Actual methods forpreparing parenterally, orally, and topically administrable compoundswill be known or apparent to those skilled in the art and are describedin detail in, for example, Remington's Pharmaceutical Science, 17th ed.,Mack Publishing Company, Easton, Pa. (1985), which in incorporatedherein by reference.

[0041] Determination of an effective amount of peptide to treat hostsafflicted with different ailments may be determined through standardempirical methods which are well known in the art. For example,immunomodulation may be monitored by serial determinations of leukocytecount, sheep red blood cell erythrocyting activity, determination ofrelative and absolute levels of different leukocyte subsets (e.g., CD4and CD8 subsets of T lymphocytes), sedimentation rates, C-reactiveprotein levels, immunoglobulin levels (particularly those directed atself-antigens), complement levels, and like, as well as general organfunction of the host. Anemias may be monitored by serial determinationsof hematocrit, hemoglobin, mean corpuscular volume, and the like. Wellknown methods of monitoring the treatment of infection include, e.g.,culture and organ function indices. Atopic states may be evaluated bychallenges to allergens and determination of IgE levels. Leukocyticdisorders may be monitored by determination of white blood cell countsand leukocyte function assays. Vaccine augmentation may be monitored byrepeated challenge of antigen, either virulent or attenuated, andobservation of the host's immune response to the challenge.

[0042] Compositions of the invention are administered to a host alreadysuffering from an infection, as described above, in an amount sufficientto cure or at least partially arrest the disease and its complications.An amount adequate to accomplish this is defined as “therapeuticallyeffective dose.” Amounts effective for this use will depend on theseverity of the infection or disease and the weight and general state ofthe patient being treated, but generally range from about 0.001 mg/kg toabout 5000 mg/kg host body weight of peptide per day, more commonlyabout 0.1 mg/kg to about 1000 mg/kg host body weight of peptide per day,usually about 0.25 mg/kg to about 100 mg/kg host body per day, moreusually about 0.5 mg/kg to about 20 mg/kg host body weight per day, andpreferably about 0.7 mg/kg to about 10 mg/kg host body weight per day.Maintenance dosages over a prolonged period of time may be adjusted asnecessary. It must be kept in mind that the materials of the presentinvention may be employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, in view of the minimization of extraneous substances and generallack of immunogenicity when a human-derived polypeptide is employed totreat human hosts, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of thesecompositions. For veterinary uses higher levels may be administered asnecessary while avoiding, however, undesirable toxicities.

[0043] In prophylactic applications, compositions containing the presentinvention are administered to a patient susceptible to or otherwise atrisk for infection, anemia, or other disorder that may be treated by themethods of the present invention. Such an amount is defined to be a“prophylactically effective dose.” In this use, the precise amountsagain depend on the patient's state of health and weight, but aregenerally in the ranges described above for therapeutic use.Prophylactic administration may be particularly desirable for hosts thathave been exposed or at risk for exposure of infectious diseases, e.g.,health-care workers, travellers, family members of infected individuals,immunosuppressed persons, and the like. The peptides of the presentinvention may also be administered for surgical prophylaxis to lessenthe risk of infectious complications and enhance the host's restorativeresponse to blood loss.

[0044] Single or multiple administrations of the compositions can becarried out with the dose levels and pattern being selected by thetreating physician or veterinarian. In any event, the pharmaceuticalpreparations should provide a quantity of sufficient to effectivelytreat, prevent, or inhibit disease in the host.

[0045] For the treatment of infection, the pharmaceutical preparationsof the present invention may be administered alone or as adjuncttherapy. The compositions may be administered with, e.g., antibiotics,anti-viral compounds, anti-fungal compounds, and anti-parasiticcompounds. When employed to enhance a host's immune response to a tumorthrough immunomodulation, the peptides of the present invention may beadministered with a variety of compounds for the treatment ofmalignancy. When administered as adjunct therapy, the compositions ofthe present invention may be administered in conjunction with the othertreatment modalities, or separately at different intervals.

[0046] The peptides of the present invention may be synthesized by avariety of techniques well known in the art. Generally, the peptideswill be prepared in solution or on a solid support by conventionalpeptide synthesis, including the Merrifield solid state peptidesynthesis technique. For example, an amino and side chain protectedderivative of an activated ester of Glx is reacted with side-groupprotected L-Lys, attached to the solid phase as its C-terminus. Afterelimination of the alpha-amino protecting group, the next amino acid isadded in a similar fashion. Additional amino acids are serially added.The peptides are cleaved by highly acidic cleavage that also typicallyremoves protecting groups. The peptides may then be isolated andlyophilized and stored for future use. Suitable techniques of peptidesynthesis are described in detail in Stewart and Young, Solid PhasePeptide Synthesis, 2d edition, Pierce Chemical Company, 1984; and Tam etal., J. Am. Chem. Soc., 105:6442 (1983), both of which are incorporatedherein by reference.

[0047] Alternatively, hybrid DNA technology may be employed forexpression of the desired peptide in transformed eukaryotic orprokaryotic host cells. See, for example, Maniatis et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982,incorporated herein by reference.

[0048] The present invention provides methods of using the compositionsand preparations of the present invention. The methods generallycomprise administering to the host a peptide having the formulaR′-Glx-Lys-R″ or a pharmaceutically acceptable salt thereof, wherein Glxis Glu or Gln; R′ is H- or a first amino acid sequence having fewer than7 amino acids; R″ is -H or a second amino acid sequence having fewerthan 7 amino acids; and the peptide has a sequence of at least 2 and notmore than 9 amino acids. A therapeutic or prophylactic amount of thepeptide will typically be administered. Generally either Glx-Lys andThr-Ala-Glx-Glx-Lys will be employed in the claimed methods althoughother peptides may be used.

[0049] The method described above may be employed to modulate theactivity of a host's immune system. The activity of the immune systemmay be enhanced or suppressed by the claimed methods. Immunologicalenhancement may occur following administration of the peptides toimmunosuppressed or immunodeficient hosts. This may be employed as atreatment for a variety of primary disease states as well as secondaryeffects of diseases or treatments. For example, primaryimmunodeficiencies, such as the acquired immunodeficiency syndrome(AIDS), DeGeorge's syndrome, severe combined immunodeficiency, and thelike, may be treated by these methods. Secondary immunodeficiencies,such as anergy from tuberculosis, drug-induced leukopenia, non-HIV viralillnesses leukopenia, radiation poisoning, toxin exposure, malnutrition,and the like, may be treated by immunomodulating the activity of ahost's immune system. Enhancement of the host's immune system may aid inthe therapy of a variety of diseases including, e.g., malignancies,infections, and the like.

[0050] Immunomodulation by the methods of the present invention may alsobe employed for the treatment of hyperactive immune states. Suchhyperactive immune states include, e.g., systemic lupus erythematosis,rheumatic fever, rheumatoid arthritis, multiple sclerosis, and the like.Treatment with peptides as described above may restore the naturalbalance to the immune system and lessen or eliminate the immunologicalreaction to host tissue.

[0051] The methods may also be employed for the treatment of a varietyof infections. These infections include bacterial infections, viralinfections, fungal infections, and parasitic infections. Typicalbacterial infections that may be treated by the methods of the presentinvention include, e.g., mycobacterial infections (e.g., tuberculosis,leprosy, M. aviurn, M. intracellulare), gram positive infections (e.g.,Staphylococus, Streptococcus, and Listeria), gram negative infections(e.g., Pseudomonas), mycoplasma, spirochetal infections (e.g.,syphilis), and the like. Both aerobic and anaerobic bacteria may betreated by the methods of the present invention.

[0052] Viral infections that may be treated by the methods of thepresent invention include HIV-1 and HIV-2, cytomegalovirus, herpessimplex virus Type I and Type II, Epstein-Barr virus, HTLV-I andHTLV-II, Marek's disease, hog cholera virus, feline sarcoma virus,distemper virus, and the like. Fungal infections, such as, e.g., Candidaalbicans, Histoplasmosis, Coccidiomycosis, Aspergillosis, andCryptococcus may be treated by the methods of the present invention.Parasitic diseases including, e.g., malaria, schistosomiasis,toxoplasmosis, leishmaniasis, and pneumocystis, may be treated by themethods of the present invention.

[0053] As described above, the methods for treating infections willoften include administration of an appropriate antibiotic, anti-viralcompound, anti-fungal compound, or anti-parasitic compound. Persons ofskill will readily appreciate how to make the selection of appropriateadjuvant therapy. Often, the methods of the present invention will allowfor reduction of the dose of the adjuvant anti-infective employed. Thismay serve as a means to reduce or eliminate dose-related complicationsand side effects.

[0054] The methods of the present invention may also be used to treatatopic states. The peptides described above may modulate thosecomponents of the immune system responsible for allergic reactions. Thismay provide an effective treatment for diseases such as acute allergicreactions, chronic urticaria, and the like.

[0055] A variety of leukocytic disorders may also be treated by themethods of the present invention. Such disorders include pre-leukemias,leukemoid reactions, and the like. A variety of anemias may be treatedby the methods of the present invention. These anemias include, acutehemorrhagic anemia, anemias of chronic disease, megaloblastic anemias,iron deficiency anemias, hemoglobinopathies, and the like.

[0056] The methods of the present invention may also indirectly enhancewound healing. This may occur by reducing local inflammation in thewound site and decreasing the risk of infection.

[0057] The following examples are offered by way of illustration and notlimitation.

EXAMPLE 1 Effect of Immune System of Health Guinea Pigs

[0058] Forty male guinea pigs were used in the following test. Most ofthe animals were treated daily with a single dose (i.m.) ofThr-Ala-Glu-Glu-Lys (HN897) of microgram/kg for five days. Controlanimals were treated with single daily doses of 0.5 ml (i.m.) of normalsaline.

[0059] Tested parameters: clinical blood examination, non-specificresistance by lysosomal cationic test; “active” T-lymphocytes and totalT-lymphocytes (E-RFC), B-lymphocytes (EAC-RFC) were measured in blood,thymus, lymph nodes, spleen and red bone marrow. Blood lymphocytefunctional activity was evaluated by leukocyte migration inhibition(LMI) with ConA. Histological examinations of thymus, lymph nodes,spleen and red bone marrow. Blood lymphocyte functional activity wasevaluated by leukocyte migration inhibition (LMI) with ConA.Histological examinations of thymus, spleen, lymph nodes, bone marrowand adrenals were carried out. All of these indicia were measured on10th and 20th days after onset of the treatment.

[0060] Principal findings: the peptide stimulates lymphoid cellsproliferation and differentiation in thymus and bone marrow; on the 10thday the predominant T-lymphocyte stimulation is observed, on the 20th-both T- and B-lymphocytes. On the 10th day after onset ofadministration, the peptide causes increased level of mitotic activityin lymph notes, spleen, and especially bone marrow.

EXAMPLE 2 Effect on Immune System

[0061] Twenty-two male guinea pigs were used in the following test.

[0062] Guinea pigs were exposed to irradiation in a total does1Gy-target-skin distance—70 cm; time of exposure—2′48″ Device: 180 kV;15 mA; filter 0.5 Cu Treatment: i.m. single daily 1 microg. HM897 per kgfor 5 days.

[0063] Treatment of controls: normal saline 0.5 ml i.m. single daily for5 days.

[0064] Leukocyte and lymphocyte levels were measured in peripheral bloodon the 7th, 14th, 21st, 36th and 44th day after irradiation.

[0065] Principal findings: the peptide stimulated proliferation of bloodlymphoid cells resulted in restoration of leukocyte and lymphocytelevels. In controls there were no immune cells restoration during allperiod of observation.

[0066] In a second test 40 male guinea pigs were used, and the sameregimen was followed.

[0067] There were two controls—irradiated and non-irradiated. Parameterswere evaluated on the 8th and 21st days after irradiation. Testedparameters: clinical blood examination, non-specific resistance bylysosomal cationic test; “active” T-lymphocytes and total T-lymphocytes(E-RFC), B-lymphocytes (EAC-RFC) were measured in blood, thymus, lymphnodes, spleen and red bone marrow. Blood lymphocyte functional activitywas evaluated by leukocyte migration inhibition (LMI) with ConA.Histological examinations of thymus, spleen, lymph nodes, bone marrowand adrenals were carried out.

[0068] Principal findings: the peptide used in irradiated animalsaccelerates T-lymphocyte maturation and their migration to peripheralimmune organs in early terms of observation. In the later stage of thestudy effects were more pronounced in the enhancement of proliferationand differentiation in both central and peripheral organs of the immunesystem. Administration restored peripheral blood lymphocytes andneutrophil functional activity.

EXAMPLE 3 Effects in Thyxnectomized Guinea Pigs

[0069] Model: Thymectomy (removal of thymus): 30 mongrel male guineapigs.

[0070] Treatment: i.m. single daily 1 microg HN897 per kg for 10 days.

[0071] Treatment of controls: normal saline 0.5 ml i.m. single daily for10 days (there were two controls -thymectomized and sham-operated).

[0072] Parameters were determined on the 15th day after onset of thetreatment.

[0073] Tested parameters: clinical blood examination, “active”T-lymphocytes and total T-lymphocytes (E-RFC), B-lymphocytes (EAC-RFC)were measured in blood, thymus, lymph nodes, spleen and red bone marrow.

[0074] Principal findings: the peptide use in thymectomized animals doesnot stimulate lymphoid cells differentiation, but, on the contrary, doessuppress it to some degree.

EXAMPLE 4 Effect on Superficial Receptors Expression on T- andB-Lymphocytes

[0075] Model: A. This work was designed to study the restoration ofsuperficial receptors on lymphocytes after proteolytic digestion orafter severe secondary immunodeficiency. Thymocytes obtained fromguinea-pig were trypsinized and then their rosette-forming capacity withrabbit erythrocytes (E-RFC) was evaluated. The cells were incubated withthe peptide in concentrations 1, 10 and 100 microg/mi. There were twocontrols—intact thymocytes (not trypsinized) and trypsinized thymocytesnot incubated with the peptide.

[0076] Principal findings: The peptide was the most active inconcentration 10 microg/mi—its biological activity made up 78/9%(percentage of rosette-forming capacity restoration).

[0077] B: B-lymphocytes were obtained from patients with streptococcaland staphylococcal skin disease showed highly pronounced secondaryimmunodeficiency. The number of cells carrying Ig-receptors before andafter incubation with the peptide has been measured (by means forFITC-labelled sera against human Ig).

[0078] Principal findings: the peptide in concentration 1 microg/mlcauses significant increase of cells carrying Ig-receptors of differenttypes.

EXAMPLE 5 Erythropoietic Effects

[0079] This test was designed to study posthemorrhagic anemia (acuteblood loss caused by taking blood from retroorbital sinus), andhemolytic anemia induced by phenylhydrazine hydrochloride (120 mg activeingredients/kg 30 Balb/c-mice and 30 CBA-mice).

[0080] HM897 was injected intraperitoneally in doses of 100 and 150microg per kg, 3 hours and 1 day after intervention modelling anemia,for 5 days.

[0081] Tester parameters: RBC, leukocytes, reticulocytes, Hb, Hct

[0082] Principal findings: 1. in posthemorrhagic anemia the mostpronounced alternations of tested parameters were observed on 4th-5thday after the invasion: erythrocytes dropped to 4.2 mln/ml vs. 6.2mln/ml in control, reticulocytes, rose 3 times, leukocytes were alsoincreased. After hg administration, on the 6th day, RBC count rose up to7.1 mln/ml; Hb level and plasma/formed elements ration restoration weremore rapid. The peptide was the most effective in dose 150 microg/kg.

[0083] 2. In hemolytic phenylhydrazine-induced anemia the mostpronounced hemodepression has arisen on the 7th day. RBC dropped to 3.8mln/ml, Hb was diminished by 15%, reticulocytes have grown up to 15%. Onthe 3rd day of administration erythrocytes have increased up to 7.2mm/ml and remained on this level in later terms. Thus, the peptide haserythropoietic effect in anemias of different genesis.

EXAMPLE 6 Influence on Colony-Forming Activity

[0084] This test is designed to study macrophage precursors. Culturedcells used were guinea-pig myelokaryocytes. HM897 was added to cellculture in concentrations 1.0, 0.001, 0.00001 and 0.0000001 microg/ml.

[0085] The peptide stimulates macrophage precursors colony-formingactivity in concentration starting form 0.0000001 microg/ml.

EXAMPLE 7 Hemostimulating Effect

[0086] The test was designed to study hemodepression induced by5-fluorouracil injected i.p. in a dose 175 mg per kg (172 maleCBA-mice). Treatment: peptide HM897 was administered i.p. starting from4th day after 5-FU injection in doses 0.00001, 0.001, 0.01, 1.0 mg/kgfor 5 days.

[0087] Treatment of controls: normal saline i.p. for 5 days.

[0088] Tested parameters: peripheral blood count and bone marrowdifferential count.

[0089] Principal findings: The peptide use promotes active restorationof hemopoiesis. This resulted in normalization of leukocytes and all CBCparameters. In bone marrow the peptide causes restoration of cellularitynormalization of all lines of hemopoiesis. The peptide was activestarting form 0.001 mg/kg.

EXAMPLE 8

[0090] This example demonstrates the effectiveness of the pharmaceuticalpreparations containing the peptide Glu-Lys (EK) for the treatment ofinfection. Mice inoculated with lethal doses of methicillin resistantStaphylococcus aureus were shown to have markedly enhanced survival whentreated with the pharmaceutical preparation.

[0091] Animals were inoculated intraperitoneally with 10×LD50 of aStaphylococcus aureus suspended in brain-heart infusion broth containing5 percent mucin. Ampicillin was administered s.c., i.p., or p.o., onehour following bacterial inoculation and deaths occurring during thesubsequent three days are recorded. The infecting organisms wereAmpicillin-resistant. If significant prevention of mortality (>50percent survival) was observed, the minimum effective dose (MED) wasdetermined.

[0092] Previous studies using methicillin-resistant S. aureus (MR)revealed that the MED for Ampicillin is much greater than 100 mg/kg fors.c. administration in the hour subsequent to inoculation.

[0093] Two control experiments were initially conducted. EK wasadministered as a pretreatment prior to microbial inoculation. Noantibiotic was administered to this group. The number of survivors at 72hours was determined. In the second control experiment, saline wasadministered in the pretreatment regimen, and Ampicillin wasadministered i.p., at the MED, in the hour following inoculation withthe microbe. No EK was administered. The number of survivors at 72 hourswas recorded. The influence of EK on survival in the presence ofAmpicillin, and in the absence of Ampicillin, after administration of S.aureus was determined by conducting experiments in the following manner.

[0094] EK was administered in a pretreatment regimen protocol to allanimals except the control groups receiving saline alone or ampicillinand saline. The influence of EK was determined over a range ofconcentrations of 10, 100, and 1000 μg/kg with single daily i.p.administrations for 3 days after which the mice were administered thenormally lethal 10×LD50 S. aureus. Survival statistics were determinedover the periods of 12, 24, 36, 48, and 72 hours. The potentialsynergistic activity between EK and Ampicillin was determined byadministering EK in the pretreatment period, and then 1 hour afterinoculum administering Ampicillin. Survival statistics were determinedover the periods 12, 23, 36, 48, and 72 hours. FIG. 1 illustrates theexperimental protocol.

[0095] Intraperitoneal administration of S. aureus at 10 times the LD50resulted in rapid deterioration of animals due to acute peritonitis. Allanimals in the control group receiving saline alone perished, and theefficacy of EK was determined by comparisons of groups treated with EKto controls receiving saline alone or Ampicillin and saline.

[0096] Bacterial culture experiments revealed that EK provides noinhibitory nor suppressive activity on microbial growth over the rangeof concentrations exceeding 1000 times the administered dose to animals.

[0097] The results are summarized in Tables 1-5. Animals administered EKwere protected. In the control group there were no survivors at 24hours. In marked contrast, as many as 78% of the EK treated groupssurvived at 36 hours. The effect of co-administration of Ampicillinresulted in at least 88% survival at 36 hours compared to only 28%survival if Ampicillin was administered alone. FIG. 2 illustrates thisdata. By 72 hours at least 27% of the animals receiving only EK werealive, and at least 77% were alive if treated with both Ampicillin andEK.

[0098] Administration of EK to mice who are subsequently inoculated witha normally lethal i.p. dose of S. aureus results in a dramaticimprovement in survival. Moreover, the administration of Ampicillin tomice pre-treated with EK results in a further 3-fold increase insurvival as compared to either treatment with Ampicillin or EK alone. Ifneither peptide or Ampicillin was administered, all animals died. Theseresults are summarized in Tables 1-5 below. FIGS. 3 and 4 graphicallyillustrate the efficacy of treatment of infections with EK. TABLE 1Survival Statistics 12 HOURS After Inoculation with S. aureus Chi-spuarestatistics Test Dose Survival compared to: article (mcg/kg) Total DeathsSurvivors (%) Control AA CD Control — 18 4 14 77.78 — — — EK 10 18 0 18100.00 P<0.05 n/s — EK 100 18 2 16 88.89 n/s n/s — EK 1000 18 1 17 94.44n/s n/s — Amp MED 18 0 18 100.00 P<0.05 — — EK Amp 10 18 0 18 100.00P<0.05 n/s n/s EK Amp 100 18 0 18 100.00 P<0.05 n/s n/s EK Amp 1000 18 018 100.0 P<0.05 n/s n/s

[0099] TABLE 2 Survival Statistics 24 HOURS After Inoculation with S.aureus Chi-spuare statistics Test Dose Survival compared to: article(mcg/kg) Total Deaths Survivors (%) Control AA CD Control — 18 18 0 0.00— — — EK 10 18 5 13 72.22 P<0.001 n/s — EK 100 18 5 13 72.22 P<0.001 n/s— EK 1000 18 1 17 94.44 P<0.001 P<0.05 — Amp MED 18 9 9 50.00 P<0.05 — —EK Amp 10 18 1 17 94.44 P<0.001 P<0.05 n/s EK Amp 100 18 1 17 94.44P<0.001 P<0.05 n/s EK Amp 1000 18 0 18 100.00 P<0.001 P<0.05 n/s

[0100] TABLE 3 Survival Statistics 36 HOURS After Inoculation with S.aureus Chi-spuare statistics Test Dose Survival compared to: article(mcg/kg) Total Deaths Survivors (%) Control AA CD Control — 18 18 0 0.00— — — EK 10 18 9 9 50.00 P<0.05 n/s — EK 100 18 9 9 50.00 P<0.05 n/s —EK 1000 18 4 14 77.78 P<0.001 P<0.05 — Amp MED 18 13 5 27.78 P<0.05 — —EK Amp 10 18 2 16 88.89 P<0.001 P<0.001 P<0.05 EK Amp 100 18 2 16 88.89P<0.001 P<0.001 P<0.05 EK Amp 1000 18 0 18 100.00 P<0.001 P<0.001 P<0.05

[0101] TABLE 4 Survival Statistics 48 HOURS After Inoculation with S.aureus Chi-spuare statistics Test Dose Survival compared to: article(mcg/kg) Total Deaths Survivors (%) Control AA CD Control — 18 18 0 0.00— — — EK 10 18 13 5 27.78 P<0.05 n/s — EK 100 18 13 5 27.78 P<0.05 n/s —EK 1000 18 10 8 44.44 P<0.05 n/s — Amp MED 18 13 5 27.78 P<0.05 — — EKAmp 10 18 4 14 77.78 P<0.001 P<0.05 P<0.05 EK Amp 100 18 4 14 77.78P<0.001 P<0.05 P<0.05 EK Amp 1000 18 2 16 88.89 P<0.001 P<0.001 P<0.05

[0102] Survival Statistics 72 HOURS After Inoculation with S. aureusChi-spuare statistics Test Dose Survival compared to: article (mcg/kg)Total Deaths Survivors (%) Control AA CD Control — 18 18 0 0.00 — — — EK10 18 13 5 27.78 P<0.05 n/s — EK 100 18 13 5 27.78 P<0.05 n/s — EK 100018 11 7 38.89 P<0.05 n/s — Amp MED 18 13 5 27.78 P<0.05 — — EK Amp 10 184 14 77.78 P<0.001 P<0.05 P<0.05 EK Amp 100 18 4 14 77.78 P<0.001 P<0.05P<0.05 EK Amp 1000 18 2 16 88.89 P<0.001 P<0.001 P<0.05

EXAMPLE 9

[0103] This example demonstrates the efficacy of pharmaceuticalpreparations containing the peptide Thr-Ala-Glu-Glu-Lys (HN897) for thetreatment of infection,. Mice inoculated with lethal doses ofmethicillin-resistant Staphylococcus aureus were shown to have markedlyenhanced survival when treated with the pharmaceutical preparation,although HM897 alone appears to have no measurable specific antibioticactivity (MIC values>1000) in petri culture experiments.

[0104] As in Example 8, animals were inoculated intraperitoneally with10×LD50 of a methicillin-resistant S. aureus suspended in brain-heartinfusion broth containing 5 percent mucin. Different treatments wereadministered s.c., i.p, or p.o., one hour later and deaths occurringduring the subsequent three days were recorded. If significantprevention of mortality (>50 percent survival) was observed, the minimumeffective dose (MED) was determined. As described above, in S. aureus(MR), the MED for Ampicillin is much greater than 100 mg/kg for s.c.administration in the hour subsequent to inoculation.

[0105] Two control experiments were conducted. HM897 was administered asa pretreatment prior to microbial inoculation, and saline wasadministered i.p. The number of survivors at 72 hours was determined. Inthe second control experiment, saline was administered in thepretreatment regimen, and Ampicillin was administered i.p., at the MED,in the hour following inoculation with microbe. The number of survivorsat 72 hours was recorded. The influence of HM897 on survival in thepresence of Ampicillin, and in the absence of Ampicillin, afteradministration of S. aureus (MR), was determined as follows.

[0106] HM897 was administered in a pretreatment regimen protocol to allanimals except the control groups provided with either saline alone orAmpicillin and saline. The effect of HM897 treatment was determined overa range of concentrations of 10, 100, and 1000 μg/kg with single dailyi.p. administrations for 3 days after which the mice were administeredthe normally lethal 10×LD50 S. aureus. Survival statistics weredetermined over the periods of 12, 24, 36, 48, and 72 hours. Thepotential synergistic activity between HM897 and ampicillin wasdetermined by administering HM897 in the pretreatment period, andadministering ampicillin 1 hour following the microbial inoculum.Survival statistics were determined over periods of 12, 23, 36, 48, and72 hours. The method of conducting the experiment is illustrated in FIG.5.

[0107] Administration of 10 times the LD50 inoculum resulted in rapiddeterioration of animals due to acute peritonitis. All animals in thecontrol group receiving saline alone died and the efficacy of HM897treatment was determined by comparisons of HM897-treated groups tosaline and ampicillin treated control groups in which no HM897 wasadministered.

[0108] The results of the experiment are summarized in Tables 6-10below. Animals receiving HM897 exhibited increased survival. In thecontrol group receiving saline alone, there were less than 6% survivorsby 24 hours, and no survivors by 36 hours. As many of 61 percent of theHM897-treated groups at 36 hours. The effect of co-administration ofAmpicillin resulted in 77% to 94% survival at 36 hours compared to only28% survival in the group receiving ampicillin alone. By 72 hours 22-39%of the animals receiving HM897 alone were alive, and 72-94% survived iftreated with ampicillin and HM897.

[0109] Administration of HM897 to mice that are subsequently inoculatedwith a normally lethal i.p. dose of S. aureus provided a dramaticimprovement in survival. Moreover, the administration of ampicillin tomice pre-treated with HM897 resulted in a further 2-fold increase insurvival as compared to either Ampicillin or HM897 administration alone.If neither HM897 or ampicillin were administered, all animals died.FIGS. 6-8 below. Comparison of FIGS. 7 and 8 reveal dramatic differencesin survival for mice that received co-administration of Ampicillin in asingle dose accompanying HM897 administration. This demonstrates adramatic synergistic effect between HM897 and ampicillin. TABLE 6Survival Statistics 12 HOURS After Inoculation with S. aureus Chi-spuarestatistics Test Dose Survival compared to: article (mcg/kg) Total DeathsSurvivors (%) Control AA CD Control — 18 4 14 77.78 — — — HM897 10 18 018 100.00 P<0.05 n/s — HM897 100 18 0 18 100.00 P<0.05 n/s — HM897 100018 1 17 94.44 n/s n/s — Amp MED 18 0 18 100.00 P<0.05 — — HM897 Amp 1018 0 18 100.00 P<0.05 n/s n/s HM897 Amp 100 18 0 18 100.00 P<0.05 n/sn/s HM897 Amp 1000 18 1 178 94.44 n/s n/s n/s

[0110] TABLE 7 Survival Statistics 24 HOURS After Inoculation with S.aureus Chi-spuare statistics Test Dose Survival compared to: article(mcg/kg) Total Deaths Survivors (%) Control AA CD Control — 18 17 1 5.56— — — HM897 10 18 5 13 72.22 P<0.001 n/s — HM897 100 18 3 15 83.33P<0.001 P<0.05 — HM897 1000 18 2 16 88.89 P<0.001 P<0.05 — Amp MED 18 99 50.00 P<0.05 — — HM897 Amp 10 18 2 16 88.89 P<0.001 P<0.05 n/s HM897Amp 100 18 1 17 94.44 P<0.001 P<0.05 n/s HM897 Amp 1000 18 1 17 94.44P<0.001 P<0.05 n/s

[0111] TABLE 8 Survival Statistics 36 HOURS After Innoculation with S.aureus Chi-square statistics Test Dose Survival compared to: article(mcg/kg) Total Deaths Survivors (%) Control AA CD Control — 18 18  0 0.00 — — — HM897  10 18 12  6 33.33 P <0.001 n/s — HM897  100 18  8 1055.56 P <0.001 n/s — HM897 1000 18  7 11 61.11 P <0.001 P <0.05  — AmpMED 18 13  5 27.78 P <0.05  — — HM897  10 18  4 14 77.78 P <0.001 P<0.05  P <0.05 Amp HM897  100 18  1 17 94.44 P <0.001 P <0.001 P <0.05Amp HM897 1000 18  2 16 88.89 P <0.001 P <0.001 n/s Amp

[0112] TABLE 9 Survival Statistics 48 HOURS After Inoculation with S.aureus Chi-square statistics compared Test Dose Survival to: article(mcg/kg) Total Deaths Survivors (%) Control AA CD Control — 18 18  0 0.00 — — — HM897  10 18 14  4 22.22 P <0.05  n/s — HM897  100 18 12  633.33 P <0.05  n/s — HM897 1000 18 11  7 38.89 P <0.05  n/s — Amp MED 1813  5 27.78 P <0.05  — — HM897  10 18  5 13 72.22 P <0.001 P <0.05  P<0.05  Amp HM897  100 18  1 17 94.44 P <0.001 P <0.001 P <0.001 AmpHM897 1000 18  3 15 83.33 P <0.001 P <0.05  P <0.05  Amp

[0113] TABLE 10 Survival Statistics 72 HOURS After Inoculation with S.aureus Chi-square statistics compared Test Dose Survival to: article(mcg/kg) Total Deaths Survivors (%) Control AA CD Control — 18 18  0 0.00 — — — HM897  10 18 14  4 22.22 P <0.05  n/s — HM897  100 18 12  633.33 P <0.05  n/s — HM897 1000 18 11  7 38.89 P <0.05 n/s — Amp MED 1813  5 27.78 P <0.05  — — HM897  10 18  5 13 72.22 P <0.001 P <0.05  P<0.05  Amp HM897  100 18  1 17 94.44 P <0.001 P <0.001 P <0.001 AmpHM897 1000 18  3 15 83.33 P <0.001 P <0.05  P <0.05  Amp

EXAMPLE 10

[0114] This example demonstrates the augmentation of a vaccine byadministration of a peptide Thr-Ala-Glu-Glu-Lys (HM897). Administrationof HM897 following vaccination provided enhanced protection againstinfection.

[0115] The present investigation was performed in hatchling fish. Inthese animals, the lymphoid system is poorly developed, withdemonstrable immune incompetence. Hatchlings exposed to certain antigensprior to maturation, instead of responding by producing antibodies, areblocked with the induction of “negative” immunity, failing tosubsequently respond to antigen stimulation. The effects of suchblocking may last several weeks, with a subsequent loss of memoryresulting in more mature fish that are highly susceptible to futureinfection.

[0116] A number of non-specific factors have been observed in the fishova, including C-reactive protein-like precipitins, and lecithin-likeagglutinins, and some fish immunoglobulins (Ig) suggesting that passiveimmunity may be provided by the parent in some species of fish. Passiveimmunity has not been observed in salmonids.

[0117] The major lymphoid organs in teleost fish are the thymus, kidney,and spleen. The thymus is composed of developing lymphocytes, and as inother vertebrates, it is regarded as the pool of virgin lymphocyteswhich subsequently emigrate to join the pool of peripheral cells of thecirculation and lymphoid organs.

[0118] The thymus is the first lymphoid organ to develop lymphocytes inhatchlings, and in rainbow trout, the fully differentiated thymus isseparated from the external environment only by a single layer ofepithelial cells, which possess pores up to 20 μm in diameter. Theseepithelial fenestrations are observed to close in older fish.

[0119] Lymphoid cells first appear in the blood and lymphoid troutwithin 3 days of hatching. The rate of growth of the lymphoid tissuesexceeds the rest of the body's rate of growth in the first few weeks oflife. The weight of the lymphoid organs relative to the body weightteaches a peak at 2 months of age when the trout are normally 0.5 grams;thereafter the relative weights diminish. An intense period of mitoticactivity occurs within the thymus during these first few months. Thymicinvolution appears to occur after approximately 9 months.

[0120] Morphologically identifiable lymphocytes are seen in earlyhatchlings, and the T- and B-lymphocytes develop at different speeds.The majority of cells have no surface immunoglobulins prior to 48 days,and the ability to produce antibody prior to this age is unlikely. Thissuggests that B-cells and T-suppressors become functionally active atabout 4 weeks of age, and T-helper cell functions mature later at about8 weeks of age.

[0121] In the present investigation, fish were obtained and inoculatedat approximately 12 weeks of age. Immunologic studies were conductedunder a variety of conditions in which fish were provided withvaccination to observe vaccine augmentation, and under conditions inwhich no vaccine was provided prior to inoculum. The effects of exposuretime, HM897 concentration, and vaccine on mortalities relative tocontrol studies in which no HM897 was administered provided data foranalysis of the immunologic effect of HM897.

[0122] A study was carried out by Bio-Research Laboratories (BRL) todetermine if HM897 possesses immune enhancing properties using a fishdisease model. The study was conducted using Rainbow trout (Oncorhynchusmykiss) to compare the effects of a standard fish vaccine to theprotection provided by varying dosages of HM897. The fish werechallenged with Vibrio anguillarum (V-775), a bacteria that causes fatalvibriosis, typically 3-5 days following pathogen exposure

[0123] Rainbow Trout (Oncorhynchus mykiss) weighing approximately 5grams were used as test organisms. The fish were obtained from Cran-MarTrout Farm. Upon arrival at BRL's facility, the fish were quarantinedfor a minimum of seven days, and observed to be disease free. Duringthis period, the fish were held at a stable temperature. There wasconsistent water quality in an aerated flow-through system with a flowrate of approximately 30 liters per hour. During the holding period thefish were observed daily for signs of disease, stress, injury,hemorrhaging, and external parasite. The fish were not fed 48 hoursprior to testing.

[0124] BRL's facilities included an area for holding and acclimatingfish while providing a constant temperature of (16±2° C.) using carbonfiltered tap water. The air used for aeration was free of oil and fumes.The test fish were shielded from any disturbances. The facility was wellventilated and free of fumes. There was a 16-hour light and 8-hour darkphotoperiod. Special care was taken to prevent contamination of the fishholding tanks.

[0125] The test holding chambers were located in a special room with thetemperature remaining constant at 12±1° C. Each test chamber was11″×21″×10″. The test chambers were cleaned thoroughly before using. Astandard cleaning procedure was followed. Detergent or acetone was usedto remove organic compounds; 5% concentration of nitric acid was used toremove metals and bases; and 200 mg hypochlorite/L was used fordisinfection. Finally, the test chambers were rinsed with dilution waterbefore the start of the test.

[0126] The Rainbow trout used in this study weighed an average of 5grams.

[0127] The fish were divided into 21 groups with 50 fish per group. Eachgroup of 50 fish was maintained in twa 10-gallon tanks (25 fish/tank)

[0128] The dosage level for the challenge bacteria Vibrio anguillarum(V-775) was determined by running multiple levels of bacterial dilutionsdesigned to kill 25 to 100% of unvaccinated fish (LD25 to LD100). Thelevel of bacterial dilution to achieve a LD75 (the level designed tokill 75% of the non-vaccinated fish) was a dilution of 10⁻⁴ to 10⁻⁵. Thechallenge bacteria were grown for 48 hours at 25° C. in trypticase soybroth. The 10⁻⁴ dilution was made from this broth in 0.9% saline. Allfish excepting the control group that received no bacterial challengewere inoculated 7 days after exposure to HM897.

[0129] HM897 was provided as a powder. The following concentrations ofHM897 were made per each 38 L tank; 2.5 μg/38 L, 10 μg/38 L, 25 μg/38 L,50 μg/38 L, 75 μg/38 L, 100 μg/38 L, 150 μg/38 L, and 250 μg/38 L.

[0130] Rainbow trout hatchling were exposed to the concentration ofHM897 as described below. Same of the fish had been vaccinated whileothers had received no vaccination. In each tank, fifty vaccinated orunvaccinated fish were exposed far a period of 5 minutes to HM897, andthen removed and placed in an assigned tank according to the groupnumber.

[0131] The positive control group (III) received a vaccine made fromVibrio anguillarum (V-775). This was made through a fermentation andextraction process in the BRL laboratory facility and is equivalent tothe aquaculture industry standard vibrio vaccine.

[0132] All groups scheduled to be challenged with the Vibrio anguillarumbacterium were challenged at the LD75 dilution on day seven of theexperiment by i.p. injection of 0.1 mL of 10⁻⁴ bacterial dilution. Thefish were observed daily for mortalities.

[0133] The experimental groups were as follows:

[0134] Group I:

[0135] Fifty unvaccinated fish were placed in Group I, and observed forthree weeks. These fish did not receive treatment with HM897 and werenot challenged with bacteria.

[0136] Group II:

[0137] Fifty unvaccinated fish were exposed to 10⁻⁴ dilution of Vibrioanguillarum (V-775) on day 7 of the experiment and observed formortality. This group was not vaccinated and did not receive HM897.

[0138] Group III:

[0139] Fifty fish were vaccinated on day one and exposed to 10⁻⁴dilution of Vibrio anguillarum (V-775) on day 7 day of the experiment.The fish were not treated with HM897.

[0140] Group IVa:

[0141] On day one, fifty vaccinated fish were allowed to swim in a tankcontaining 38 liters of water with 2.5 μg of HM897 for 5 minutes andthen they were placed in the tanks marked Group IVa. On day 7 the fishwere exposed to 10⁻⁴ dilution of Vibrio anguillarum (V-775) and observedfor mortality.

[0142] Group IVb:

[0143] On day one, fifty vaccinated fish were allowed to swim in a tankcontaining 38 liters of water with 10 μg of HM897 for 5 minutes and thenthey were placed in the tanks marked Group IVb. On day 7 the fish wereexposed to 10⁻⁴ dilution of Vibrio anguillarum (V-775) and observed formortality

[0144] Group IVc:

[0145] On day one, fifty vaccinated fish were allowed to swim in a tankcontaining 38 liters of water with 50 μg of HM897 for 5 minutes and thenthey were placed in the tanks marked Group IVc. On day 7 the fish wereexposed to 10⁻⁴ dilution of Vibrio angiuillarum (V-775) and observed formortality.

[0146] Group IVd:

[0147] On day one, fifty vaccinated fish were allowed to swim in a tankcontaining 38 liters of water with 100 μg of HM897 for 5 minutes andthen they were placed in the tanks marked Group IVd. On day 7 the fishwere exposed to 10⁻⁴ dilution of Vibrio anguillarum (V-775) and observedfor mortality.

[0148] Group Va:

[0149] On day one, fifty unvaccinated fish were allowed to swim in atank containing 38 liters of water with 10 μg of HM897 for 5 minutes andthen they were placed in the tanks marked Group Va. On day 7 the fishwere exposed to 10⁻⁴ dilution of Vibrio anguillarum (V-775) and observedfor mortality

[0150] Group Vb:

[0151] On day one, fifty unvaccinated fish were allowed to swim in atank containing 38 liters of water with 25 μg of HM897 for 5 minutes andthen they were placed in the tanks marked Group Vb. On day 7 the fishwere exposed to 10⁻⁴ dilution of Vibrio anguillarum (V-775) and observedfor mortality.

[0152] Group Vc:

[0153] On day one, fifty unvaccinated fish were allowed to swim in atank containing 38 liters of water with 75 μg of HM897 for 5 minutes andthen they were placed in the tanks marked Group Vc. On day 7 the fishwere exposed to 10⁻⁴ dilution of Vibrio anguillarum (V-775) and observedfor mortality.

[0154] Group Vd:

[0155] On day one, fifty unvaccinated fish were allowed to swim in atank containing 38 liters of water with 150 μg of HM897 for 5 minutesand then they were placed in the tanks marked Group Vd. On day 7 thefish were exposed to 10⁻⁴ dilution of Vibrio anguillarum (V-775) andobserved for mortality.

[0156] Group Ve:

[0157] On day one, fifty unvaccinated fish were allowed to swim in atank containing 38 liters of water with 250 μg of HM897 for 5 minutesand then they were placed in the tanks marked Group Ve. On day 7 thefish were exposed to 10⁻⁴ dilution of Vibrio anguillarum (V-775) andobserved for mortality.

[0158] All of the groups were observed for a period of twenty-one days,and the effect of the treatments were evaluated based on the number offish mortalities. The groups are summarized in Table XI-1 below. TABLEXI-1 Summary of Experimental Design for HM897 and Retromedine ™Immunologic Studies in Rainbow Trout. Test Article Inoculated VaccinatedTest μg 38 liters LD75 Number of Group Day 1 Article Day 1 Day 7 Fish Ino none none no 50 II no none none yes 50 III yes none none yes 50 IVayes HM897 2.5 yes 50 IVb yes HM897 10 yes 50 IVc yes HM897 50 yes 50 IVdyes HM897 100 yes 50 Va no HM897 10 yes 50 Vb no HM897 25 yes 50 Vc noHM897 75 yes 50 Vd no HM897 150 yes 50 Ve no HM897 250 yes 50

[0159] The bacterial challenge (Vibrio anguillarum, V-775) dilutionlevels and daily accumulated mortalities are shown in Table XI-2. Theresults of this experiment established the LD75 of the challengebacterial. Each group of fifteen fish were challenged with bacterialdilution of 10⁻², 10⁻³, 10⁻⁴, or 10⁻⁵. Nine days after injection of thebacterial dilutions the accumulated mortalities were as follows:

[0160] At 10⁻² bacterial dilution 14 fish were dead (93.3% mortality).

[0161] At 10⁻³ bacterial dilution 13 fish were dead (86.7% morality)

[0162] At 10⁻⁴ bacterial dilution 10 fish were dead (66.7% mortality)

[0163] At 10⁻⁵ bacterial dilution 10 fish were dead (66.7% mortality).TABLE XI-2 Cumulative Mortalities for Rainbow Trout Inoculated withVibrio anguillarum LD75 Determination. Number Group Dilation of Fish06/30 07/01 07/02 07/03 07/04 07/05 07/06 07/07 07/08 07/09 1 10-2 15 00 14  14  14 14 14 14 14 14 2 10-3 15 0 0 6 9 13 13 13 13 13 13 3 10-415 0 0 0 5  9 10 10 10 10 10 4 10-5 15 0 0 0 4  7 10 10 10 10 10

[0164] The daily accumulated mortalities are shown in Table XI-3. Thenumber of the mortalities for each group were as follows:

[0165] Group I: This group had no mortalities.

[0166] Group II: Thirty-one fish died from bacterial infection (62%mortality).

[0167] Group III: Fifteen fish died from bacterial infection (30%mortality).

[0168] Group IVa: Thirteen fish died from bacterial infection (26%mortality).

[0169] Group IVb: Seven fish died from bacterial infection (14%mortality)

[0170] Group IVc: Ten fish died from bacterial infection (20% mortality)

[0171] Group IVd: Seventeen fish died from bacterial infection (34%mortality).

[0172] Group Va: Thirty-three fish died from bacterial infection (66%mortality).

[0173] Group Vb: Thirty-tree fish died from bacterial infection (66%mortality).

[0174] Group Vc: Thirty-four fish died from bacterial infection (68%mortality).

[0175] Group Vd: Thirty-two fish died from bacterial infection (64%mortality).

[0176] Group Ve: Thirty-four fish died from bacterial infection (68%mortality) TABLE XI-3 Summary Cumulative Mortalities for HM897 Influenceon Rainbow Trout Infected with Vibrio anguillarum Day Group 1 2.10 11 1213 14 15 16 17 18 19 20 21 Total Mortalities I 0 0 0 0 0 0 0 0 0 0 0 0 00 II 0 0 0 0 22 27 29 31 31 31 31 31 31 31 III 0 0 0 0 8 14 14 15 15 1515 15 15 15 IVa 0 0 0 0 1 7 10 13 13 13 13 13 13 13 IVb 0 0 0 0 1 5 6 67 7 7 7 7 7 IVc 0 0 0 0 1 5 10 10 10 10 10 10 10 10 IVd 0 0 0 0 8 15 1617 17 17 17 17 17 17 Va 0 0 0 0 16 24 28 33 33 33 33 33 33 33 Vb 0 0 0 018 25 32 33 33 33 33 33 33 33 Vc 0 0 1 1 21 29 32 33 34 34 34 34 34 34Vd 0 0 0 0 14 23 28 32 32 32 32 32 32 32 Ve 0 0 0 0 14 25 30 32 34 34 3434 34 34

[0177] TABLE XI-4 Summary of Results Trout Mortalities Test Article μgInoculated Total Percent Vaccinated Test 38 liters LD75 Mortal- Mortal-Group Day 1 Article Day 1 Day 7 ities ities I no None none no 0 0 II noNine none yes 31 62 III yes Vaccine none yes 15 30 IVa yes HM897 2.5 yes13 26 IVb yes HM897 10 yes 7 14 IVc yes HM897 50 yes 10 20 IVd yes HM897100 yes 17 34 Va no HM897 10 yes 33 66 Vb no HM897 25 yes 33 66 Vc noHM897 75 yes 34 68 Vd no HM897 150 yes 32 64 Ve no HM897 250 yes 34 68

[0178]Vibrio anguillarum (V-775) was used as challenge bacteria atto10⁻⁴ dilution. The bacterial challenge study (Table XI-2) revealedthat 10⁻⁴ and 10⁻⁵ of the bacterial dilutions can kill about 75% of theinfected fish (LD75). The 10⁻⁴ dilution was selected as a LD75 bacterialdilution.

[0179] Group I, without any treatment and with no bacterial challenge,had no mortalities. This confirmed that the fish were in good health.Adequate filtration and aeration were provided to maintain a suitableenvironment far the fish.

[0180] No mortalities in Group I and 62% mortalities in Group II clearlyindicate that all the mortalities were related to the bacterialinfection (Table XI-4). The bacterial dilution was predicted to killabout 75% of the fish, and the actual mortality of control group (GroupII) was 62%.

[0181] A typical commercial vaccine usually affords 60% to 70%protection. The vaccinated group (Group III) showed 30% mortalities andtherefore provided 70% protection against the challenge bacteria.

[0182] The fish in groups IVa, IVb, IVc, and IVd were vaccinated andthen each exposed to a different concentrations of HM897 for 5 minutes.Each group reveals a different level of protection against the challengebacteria. The fish in Group IVb with 10 μg of HM897 in 38 liters for 5minutes inoculated with challenge Vibrio anguillarum 7 days aftervaccination had the lowest mortalities at 14% compared to the groupreceiving vaccine alone at 30% (FIG. 9).

[0183] The groups treated with different levels of HM897 exposurewithout vaccine (Groups Va to Ve) showed no protection against thebacterial infection (Table XI-3, FIG. 10). The percentage of the fishmortalities in these groups were similar to the control group (Group II)in which no treatment was provided excepting challenge with Vibrioanguillarum. This indicates that HM897 has a statistically significanteffect for augmentation of vaccinations.

[0184] All publications, patents and patent applications mentioned inthis specification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

[0185] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A composition comprising a peptide having theformula R′-Glx-Glx-Lys-R″ or a pharmaceutically acceptable salt thereof;wherein Glx is Glu or Gln; R′ is H- or a first amino acid sequencehaving fewer than 7 amino acids; R″ is -H or a second amino acidsequence having fewer than 7 amino acids; and the peptide has a sequenceof at least 5 and not more than 9 amino acids.
 2. The composition ofclaim 1, wherein V is H-, Thr-Ala-, Thr-Pro-, Ser-Ala-, Ser-Pro-,Ser-Ser-, Met-Leu-Thr-Ala-, or Leu-Thr-Ala-; and R″ is -H, -Ala,-Ala-Ala or -Ala-Vat.
 3. A composition of claim 2, wherein the peptideis L-Thr-L-Pra-L-Glu-L-Glu-L-Lys.
 4. A composition of claim 2, whereinthe peptide is L-Thr-L-Ala-L-Glu-L-Glu-L-Lys.
 5. A pharmaceuticalpreparation comprising: a peptide having the formula R′-Glx-Lys-R″ or apharmaceutically acceptable salt thereof, wherein Glx is Glu or Gln; R′is H- or a first amino acid sequence having fewer than 7 amino acids; R″is -H or a second amino acid sequence having fewer than 7 amino acids;and the peptide has a sequence of at least 2 and not more than 9 aminoacids; and a physiologically acceptable carrier.
 6. The pharmaceuticalpreparation of claim 5, wherein V is H-, Glx-, Thr-Ala-Glx-,Thr-Pro-Glx-, Ser-Ala-Glx-, Ser-Pro-Glx-, Ser-Ser-Glx-,Met-Leu-Thr-Ala-Glx-, or Leu-Thr-Ala-Glx-; and R″ is -H, -Ala, -Ala-Alaor -Ala-Val.
 7. The pharmaceutical preparation of claim 6, wherein thepeptide is L-Glu-L-Lys.
 8. The pharmaceutical preparation of claim 6,wherein the peptide is L-Thr-L-Pro-L-Glu-L-Glu-L-Lys.
 9. A compositionof claim 6, wherein the peptide is L-Thr-L-Ala-L-Glu-L-Glu-L-Lys.
 10. Amethod for modulating the activity of a host's immune system, comprisingadministering to the host a peptide having the formula R′-Glx-Lys-R″ ora pharmaceutically acceptable salt thereof, wherein Glx is Glu or Gln;R′ is H-or a first amino acid sequence having fewer than 7 amino acids;R″ is -H or a second amino acid sequence having fewer than 7 aminoacids; and the peptide has a sequence of at least 2 and not more than 9amino acids.
 11. A method as in claim 10, wherein R′ is H-,Glx-Thr-Ala-Glx-, Thr-Pro-Glx-, Ser-Ala-Gix-, Ser-Pro-Glx-,Ser-Ser-Glx-, Met-Leu-Thr-Ala-Glx-, or Leu-Thr-Ala-Glx-; and R″ is -H,-Ala, -Ala-Ala or -Ala-Val.
 12. A method as in claim 11, wherein thepeptide is L-Thr-L-Ala-L-Glu-L-Glu-L-Lys or L-Glu-L-Lys.
 13. A method asin claim 10, wherein the peptide is administered in a physiologicallyacceptable carrier.
 14. A method for treating an infection in a host,comprising administering to the host a peptide having the formulaR′-Glx-Lys-R″ or a pharmaceutically acceptable salt thereof, wherein Glxis Glu or Gln; V is H- or a first amino acid sequence having fewer than7 amino acids; R″ is -H or a second amino acid sequence having fewerthan 7 amino acids; and the peptide has a sequence of at least 2 and notmare than 9 amino acids.
 15. The method as in claim 14, wherein R′ isH-, Glx-, Thr-Ala-Glx-, Thr-Pro-Glx-, Ser-Ala-Glx-, Ser-Pro-Glx-,Ser-Ser-Glx-; Met-Leu-Thr-Ala-Glx-, or Leu-Thr-Ala-Glx-; and R″ is -H,-Ala, -Ala-Ala or -Ala-Val.
 16. The method as in claim 14, wherein thepeptide is L-Thr-L-Ala-L-Glu-L-Glu-L-Lys or L-Glu-L-Lys.
 17. The methodas in claim 14, wherein the infection is a bacterial infection.
 18. Themethod as in claim 17, further comprising administering an antibiotic tothe host.
 19. The method as in claim 14, wherein the infection is aviral infection.
 20. The method as in claim 19, further comprisingadministering an anti-viral agent to the host.
 21. The method as inclaim 14, wherein the infection is a fungal infection.
 22. The method asin claim 21, further comprising administering an anti-fungal agent tothe host.
 23. The method as in claim 14, wherein the infection is aparasitic infection.
 24. The method as in claim 23, further comprisingadministering an anti-parasitic agent to the host.
 25. The method as inclaim 14, wherein the peptide is administered intravenously,intramuscularly, intrathecally, subcutaneously, intraperitoneally,intranasally, orally, intrabronchially, rectally, or topically.
 26. Amethod for treating atopic states in a host comprising administering tothe host a peptide having the formula R′-Glx-Lys-R″ or apharmaceutically acceptable salt thereof, wherein Glx is Glu or Gin; TVis H- or a first amino acid sequence having fewer than 7 amino acids; R″is -H or a second amino acid sequence having fewer than 7 amino acids;and the peptide has a sequence of at least 2 and not more than 9 aminoacids.
 27. The method as in claim 26, wherein R′ is H-, Glx-,Thr-Ala-Glx-, Thr-Pro-Glx-, Ser-Ala-Glx-, Ser-Pro-Glx-, Ser-Ser-Glx-,Met-Leu-Thr-Ala-Glx-, or Leu-Thr-Ala-Glx-; and R″ is -H, -Ala, -Ala-Alaor -Ala-Val.
 28. The method as in claim 27, wherein the peptide isL-Thr-L-Ala-L-Glu-L-Glu-L-Lys or L-Glu-L-Lys.
 29. A method of treatingleukocytic disorders in a host comprising administering to the host apeptide having the formula R′-Glx-Lys-R″ or a pharmaceuticallyacceptable salt thereof, wherein Glx is Glu or Gln; TV is H- or a firstamino acid sequence having fewer than 7 amino acids; R″ is -H or asecond amino acid sequence having fewer than 7 amino acids; and thepeptide has a sequence of at least 2 and not more than 9 amino acids.30. A method for augmenting vaccinations in a host comprisingadministering to the host a peptide having the formula R′-Glx-Lys-R″ ora pharmaceutically acceptable salt thereof, wherein Glx is Glu or Gln;R′ is H- or a first amino acid sequence having fewer than 7 amino acids;R″ is -H or a second amino acid sequence having fewer than 7 aminoacids; and the peptide has a sequence of at least 2 and not more than 9amino acids.